Establishing transgenic murine esophageal organoids

Ko, Kyung-Phil; Zhang, Jie; Park, Jae-Il

doi:10.1016/j.xpro.2022.101317

Cited by 7 publications

(6 citation statements)

References 13 publications

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“…See Supplementary Table 2 for primer information. CRISPR/Cas9-based gene KO in EOs CRISPR/Cas9 system-mediated gene KO was described in a previous study 50 . In brief, WT or Trp53 KO EOs were digested with 0.05% Trypsin-EDTA to dissociate into single cells.…”

Section: Discussionmentioning

confidence: 99%

“…Briefly, pLenti-Sox2-GFP plasmid was prepared by amplifying Sox2 template from the pLV-tetO-Sox2 plasmid(addgene, #19765), and then transferred to pLenti CMV GFP plasmid (addgene, #17446) using High-Fidelity DNA polymerase (NEB). Viral transduction was performed as described in a previous study 50 . GFP fluorescence expressing cells were isolated using flow cytometry (BD FACSAria II Cell Sorter) and cultured again.…”

Section: Sox2 Stable Cell Line Establishmentmentioning

confidence: 99%

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Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion

Huang

Zhang

et al. 2022

Preprint

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Despite recent progress in identifying aberrant genetic and epigenetic alterations in esophageal squamous cell carcinoma (ESCC), the mechanism of ESCC initiation remain unknown. Using genetically engineered esophageal organoids (EOs), we identified the key genetic determinants that drive ESCC tumorigenesis. A single-cell transcriptomic analysis uncovered that Trp53, Cdkn2a, and Notch1 (PCN) triple knockout (KO) induces neoplastic features of ESCC by generating distinct cell lineage trajectories with multiple root cells and high cell plasticity. Although Trp53 and Notch1 (PN) double KO was sufficient to induce esophageal neoplasia and cellular heterogeneity, additional inactivation of Cdkn2a was indispensable for immune landscape remodeling for in vivo tumorigenesis. PCN KO generated immunosuppressive niche enriched with exhausted T cells and M2 macrophages via the CCL2-CCR2 axis in an autochthonous ESCC mouse model. Moreover, genetic or pharmacological blockade of the CCL2-CCR2 axis suppressed ESCC tumorigenesis. Comparative single-cell transcriptomic analyses classified ESCC patient tumors into three subgroups and identified a specific subset recapitulating PCN-type ESCC signatures, including the high expression of CCL2 and CD274/PD-L1. Our study unveils that loss of TP53, CDKN2A, and NOTCH1 induces esophageal neoplasia and immune evasion for ESCC initiation and proposes the CCL2 blockade as a viable approach to target a subset of ESCC.

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Section: Discussionmentioning

confidence: 99%

Section: Sox2 Stable Cell Line Establishmentmentioning

confidence: 99%

Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion

Huang

Zhang

et al. 2022

Preprint

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“…We used Adeno-Cre virus to treat Kras LSL-G12D/+ ; Trp53 fl/fl organoids. The protocol was previously described (Ko et al, 2022; Ko et al, 2023). The cells were first dissociated from GOs as described in the organoid passaging protocol (step 1-14) .…”

Section: Methodsmentioning

confidence: 99%

CDH1 loss promotes diffuse-type gastric cancer tumorigenesis via epigenetic reprogramming and immune evasion

Zou

Huang

Zhang

et al. 2023

Preprint

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Diffuse-type gastric adenocarcinoma (DGAC) is lethal cancer often diagnosed late and resistant to therapeutics. Although hereditary DGAC is mainly characterized by mutations in the CDH1 gene encoding E-cadherin, the impact of E-cadherin inactivation on sporadic DGAC tumorigenesis remains elusive. We found that CDH1 inactivation occurs only subset of DGAC patient tumors. Unsupervised clustering of single-cell transcriptomes of DGAC patient tumors identified two subtypes of DGACs: DGAC1 and DGAC2. The DGAC1 is mainly characterized by CDH1 loss and exhibits distinct molecular signatures and aberrantly activated DGAC-related pathways. Unlike DGAC2 lacking immune cell infiltration in tumors, DGAC1 tumor is enriched with exhausted T cells. To demonstrate the role of CDH1 loss in DGAC tumorigenesis, we established a genetically engineered murine gastric organoid (GOs; Cdh1 knock-out [KO], KrasG12D, Trp53 KO [EKP]) model recapitulating human DGAC. In conjunction with KrasG12D, Trp53 KO (KP), Cdh1 KO is sufficient to induce aberrant cell plasticity, hyperplasia, accelerated tumorigenesis, and immune evasion. Additionally, EZH2 was identified as a key regulon promoting CDH1 loss-associated DGAC tumorigenesis. These findings underscore the significance of comprehending the molecular heterogeneity of DGAC and its potential implication for personalized medicine to DGAC patients with CDH1 inactivation.

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“…Fortunately, tumor organoid models have been established to study ESCC and EAC, providing valuable tools for further research in EC. 46 , 165 Ko et al 166 designed culture methods for mouse esophageal organoids and transgenic esophageal organoids using viral transduction ( Figure 9(a and c )). For EAC, organoids derived from endoscopic biopsies of patients accurately reflect the histological characteristics of the tumor.…”

Section: Classification Of Digestive Tract Tumor Organoidsmentioning

confidence: 99%

“…(a) Schematic process of murine esophageal organoid culture. Adapted from Ko et al 166 (b) Phenotypic changes in esophageal epithelium occur during normal development and Barrett’s esophagus. Adapted from Wang and Souza.…”

Section: Classification Of Digestive Tract Tumor Organoidsmentioning

confidence: 99%

Revolutionizing digestive system tumor organoids research: Exploring the potential of tumor organoids

Xiu,

Yang,

Xie

et al. 2024

J Tissue Eng

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Digestive system tumors are the leading cause of cancer-related deaths worldwide. Despite ongoing research, our understanding of their mechanisms and treatment remain inadequate. One promising tool for clinical applications is the use of gastrointestinal tract tumor organoids, which serve as an important in vitro model. Tumor organoids exhibit a genotype similar to the patient’s tumor and effectively mimic various biological processes, including tissue renewal, stem cell, and ecological niche functions, and tissue response to drugs, mutations, or injury. As such, they are valuable for drug screening, developing novel drugs, assessing patient outcomes, and supporting immunotherapy. In addition, innovative materials and techniques can be used to optimize tumor organoid culture systems. Several applications of digestive system tumor organoids have been described and have shown promising results in related aspects. In this review, we discuss the current progress, limitations, and prospects of this model for digestive system tumors.

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Establishing transgenic murine esophageal organoids

Cited by 7 publications

References 13 publications

Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion

Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion

CDH1 loss promotes diffuse-type gastric cancer tumorigenesis via epigenetic reprogramming and immune evasion

Revolutionizing digestive system tumor organoids research: Exploring the potential of tumor organoids

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